Recently, we have described a novel mechanism-based inactivation of cytochrome P-450 by a variety of drugs that appears to be mediated by radical metabolites and is characterized by the covalent attachment to the protein of products of the heme prosthetic group. In the present study We have investigated the mechanism of the formation of these adducts as well as possible biochemical effects of this modification. A model system for the reaction was developed by reacting ferrous myoglobin with bromotrichloromethane. A major heme-modified protein adduct was produced in the reaction. It was isolated, proteolytically digested, and the resulting principal peptide fragment containing a heme-derived product was characterized. Absorption and mass spectroscopy of the product revealed an intact heme moiety covalently bound to a specific N-terminal tryptophan residue. Three nonprotein bound modified hemes were also isolated from the reaction mixture. These products were characterized as a beta- carboxy substituted vinyl, an alpha-hydroxy-beta-trichloromethyl vinyl, and an alpha, beta-bis-(trichloromethyl)-vinyl derivative of the prosthetic heme by absorption, mass, and NMR spectroscopy. It is believed that the nonprotein bound heme-derived products arise from the initial attack of the trichloromethyl radical on a specific vinyl group of heme prosthetic group whereas the covalently bound product likely results from the initial attack of the trichloromethyl radical on the N-terminal tryptophan residue of the protein moiety to create a radical center, which then attacks the heme group. In addition, the heme-modified myoglobin was shown to be more susceptible to trypsin proteolysis than native myoglobin, suggesting a possible role of heme-modification in the protein turnover in vivo of cytochromes P-450 and other heme proteins.